EPA Document #: EPA/600/R-09/149 METHOD 538. … · 1.1 This is a direct aqueous injection-liquid...

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EPA Document #: EPA/600/R-09/149

METHOD 538. DETERMINATION OF SELECTED ORGANIC CONTAMINANTS

IN DRINKING WATER BY DIRECT AQUEOUS INJECTION-

LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY

(DAI-LC/MS/MS)

Version 1.0

November 2009

J.A. Shoemaker

NATIONAL EXPOSURE RESEARCH LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U. S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268

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METHOD 538

DETERMINATION OF SELECTED ORGANIC CONTAMINANTS IN DRINKING

WATER BY DIRECT AQUEOUS INJECTION-LIQUID

CHROMATOGRAPHY/ TANDEM MASS SPECTROMETRY (DAI-LC/MS/MS)

1. SCOPE AND APPLICATION

1.1 This is a direct aqueous injection-liquid chromatography/tandem mass spectrometry

(DAI-LC/MS/MS) method for the determination of selected nonvolatile chemical

contaminants in drinking water. Accuracy and precision data have been generated in

reagent water, and finished ground and surface waters for the compounds listed in the

table below.

Analyte

Chemical Abstract Services

Registry Number (CASRN)

Acephate 30560-19-1

Aldicarb 116-06-3

Aldicarb sulfoxide 1646-87-3

Dicrotophos 141-66-2

Diisopropyl methylphosphonate (DIMP) 1445-75-6

Fenamiphos sulfone 31972-44-8

Fenamiphos sulfoxide 31972-43-7

Methamidophos 10265-92-6

Oxydemeton-methyl 301-12-2

Quinoline 91-22-5

Thiofanox 39196-18-4

1.2 The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets

Data Quality Objectives (DQOs) that are developed based on the intended use of this

method. The single laboratory lowest concentration MRL (LCMRL) is the lowest true

concentration for which the future recovery is predicted to fall, with high confidence

(99%), between 50 and 150% recovery. Single laboratory LCMRLs for analytes in

this method range from 0.011-1.5 µg/L, and are listed in Table 5. The procedure used

to determine the LCMRL is described elsewhere.1

http://www.sigmaaldrich.com/catalog/Lookup.do?N5=DISPLAY_CAS&N3=mode+matchpartialmax&N4=1646-87-3&D7=0&D10=&N25=0&N1=S_ID&ST=RS&F=PR

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1.3 Laboratories using this method will not be required to determine the LCMRLs, but will

need to demonstrate that their laboratory MRL meets the requirements described in

Section 9.2.4.

1.4 Determining the Detection Limit (DL) for analytes in this method is optional (Sect.

9.2.6). Detection limit is defined as the statistically calculated minimum concentration

that can be measured with 99% confidence that the reported value is greater than zero.2

The DL is compound dependent and is dependent on sample preparation, sample

matrix, fortification concentration, and instrument performance.

1.5 This method is intended for use by analysts skilled in the operation of LC/MS/MS

instruments and the interpretation of the associated data.

1.6 METHOD FLEXIBILITY – In recognition of technological advances in analytical

systems and techniques, the laboratory is permitted to modify the separation technique,

LC column, mobile phase composition, LC conditions and MS and MS/MS conditions

(Sect. 6.6, 9.4, 10.2, and 12.1). Changes may not be made to sample collection and

preservation (Sect. 8), or to the quality control requirements (Sect. 9). Method

modifications should be considered only to improve method performance.

Modifications that are introduced in the interest of reducing cost or sample processing

time, but result in poorer method performance, should not be used. Analytes must be

adequately resolved chromatographically in order to permit the mass spectrometer to

dwell on a minimum number of compounds eluting within a retention time window.

Instrumental sensitivity (or signal-to-noise) will decrease if too many compounds are

permitted to elute within a retention time window. In all cases where method

modifications are proposed, the analyst must perform the procedures outlined in the

initial demonstration of capability (IDC, Sect. 9.2), verify that all Quality Control

(QC) acceptance criteria in this method (Sect. 9) are met, and that acceptable method

performance can be verified in a real sample matrix (Sect. 9.3.5).

NOTE: The above method flexibility section is intended as an abbreviated summation

of method flexibility. Sections 4-12 provide detailed information of specific

portions of the method that may be modified. If there is any perceived

conflict between the general method flexibility statement in Section 1.6 and

specific information in Sections 4-12, Sections 4-12 supersede Section 1.6.

2. SUMMARY OF METHOD

2.1 A 40-mL water sample is collected in a bottle containing sodium omadine and

ammonium acetate. An aliquot of the sample is placed in an autosampler vial and the

internal standards are added. A 50-µL or larger injection is made into an LC equipped

with a C18 column that is interfaced to an MS/MS operated in the electrospray

ionization (ESI) mode. The analytes are separated and identified by comparing the

acquired mass spectra and retention times to reference spectra and retention times for

calibration standards acquired under identical LC/MS/MS conditions. The

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concentration of each analyte is determined by internal standard calibration using

procedural standards.

3. DEFINITIONS

3.1 ANALYSIS BATCH – A set of samples analyzed on the same instrument, not

exceeding a 24-hour period and including no more than 20 Field Samples, beginning

and ending with the analysis of the appropriate Continuing Calibration Check (CCC)

standards. Additional CCCs may be required depending on the length of the analysis

batch and/or the number of Field Samples.

3.2 CALIBRATION STANDARD (CAL) – A solution prepared from the primary dilution

standard solution and/or stock standard solution and the internal standard(s). The CAL

solutions are used to calibrate the instrument response with respect to analyte

concentration.

3.3 COLLISIONALLY ACTIVATED DISSOCIATION (CAD) – The process of

converting the precursor ion’s translational energy into internal energy by collisions

with neutral gas molecules to bring about dissociation into product ions.

3.4 CONTINUING CALIBRATION CHECK (CCC) – A calibration standard containing

the method analytes and internal standard(s). The CCC is analyzed periodically to

verify the accuracy of the existing calibration for those analytes.

3.5 DETECTION LIMIT (DL) – The minimum concentration of an analyte that can be

identified, measured, and reported with 99% confidence that the analyte concentration

is greater than zero. This is a statistical determination of precision (Sect. 9.2.6), and

accurate quantitation is not expected at this level.2

3.6 FIELD DUPLICATES (FD1 and FD2) – Two separate samples collected at the same

time and place under identical circumstances, and treated exactly the same throughout

field and laboratory procedures. Analyses of FD1 and FD2 give a measure of the

precision associated with sample collection, preservation, and storage, as well as

laboratory procedures.

3.7 INTERNAL STANDARD (IS) – A pure chemical added to a standard solution in a

known amount(s) and used to measure the relative response of other method analytes

that are components of the same solution. The internal standard must be a chemical

that is structurally similar to the method analytes, has no potential to be present in

water samples, and is not a method analyte.

3.8 LABORATORY FORTIFIED BLANK (LFB) – A volume of reagent water or other

blank matrix to which known quantities of the method analytes and all the preservation

reagents are added in the laboratory. The LFB is analyzed exactly like a sample, and

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its purpose is to determine whether the methodology is in control, and whether the

laboratory is capable of making accurate and precise measurements.

3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – A preserved field

sample to which known quantities of the method analytes are added in the laboratory.

The LFSM is processed and analyzed exactly like a sample, and its purpose is to

determine whether the sample matrix contributes bias to the analytical results. The

background concentrations of the analytes in the sample matrix must be determined in

a separate sample and the measured values in the LFSM corrected for background

concentrations.

3.10 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A

duplicate of the Field Sample used to prepare the LFSM. The LFSMD is fortified, and

analyzed identically to the LFSM. The LFSMD is used instead of the Field Duplicate

to assess method precision when the occurrence of method analytes is low.

3.11 LABORATORY REAGENT BLANK (LRB) – An aliquot of reagent water or other

blank matrix that is treated exactly as a sample including exposure to all glassware,

equipment, solvents and reagents, sample preservatives, and internal standards that are

used in the analysis batch. The LRB is used to determine if method analytes or other

interferences are present in the laboratory environment, the reagents, or the apparatus.

3.12 LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) – The

single laboratory LCMRL is the lowest true concentration for which a future recovery

is expected, with 99% confidence, to be between 50 and 150% recovery.1

3.13 MATERIAL SAFETY DATA SHEET (MSDS) – Written information provided by

vendors concerning a chemical’s toxicity, health hazards, physical properties, fire, and

reactivity data including storage, spill, and handling precautions.

3.14 MINIMUM REPORTING LEVEL (MRL) – The minimum concentration that can be

reported as a quantitated value for a method analyte in a sample following analysis.

This defined concentration can be no lower than the concentration of the lowest

calibration standard for that analyte and can only be used if acceptable QC criteria for

this standard are met. A procedure for verifying a laboratory’s MRL is provided in

Section 9.2.4.

3.15 PRECURSOR ION – For the purpose of this method, the precursor ion is the

protonated molecule ([M+H]+) or adduct ion of the method analyte. In MS/MS, the

precursor ion is mass selected and fragmented by collisionally activated dissociation to

produce distinctive product ions of smaller m/z.

3.16 PRIMARY DILUTION STANDARD (PDS) SOLUTION – A solution containing the

analytes prepared in the laboratory from stock standard solutions and diluted as needed

to prepare calibration solutions and other needed analyte solutions.

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3.17 PRODUCT ION – For the purpose of this method, a product ion is one of the fragment

ions produced in MS/MS by collisionally activated dissociation of the precursor ion.

3.18 QUALITY CONTROL SAMPLE (QCS) – A solution of method analytes of known

concentrations that is obtained from a source external to the laboratory and different

from the source of calibration standards. The QCS is used to check calibration

standard integrity.

3.19 STOCK STANDARD SOLUTION (SSS) – A concentrated solution containing one or

more method analytes prepared in the laboratory using assayed reference materials or

purchased from a reputable commercial source.

4. INTERFERENCES

4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and tap

water, rinse with tap water, followed by a reagent water rinse. Non-volumetric

glassware can be heated in a muffle furnace at 400 °C for 2 h or solvent rinsed.

Volumetric glassware should be solvent rinsed and not be heated in an oven above

120 °C. Store clean glassware inverted, covered with foil, or capped.

4.2 Method interferences may be caused by contaminants in solvents, reagents (including

reagent water), sample bottles and caps, and other laboratory supplies or hardware that

lead to discrete artifacts and/or elevated baselines in the chromatograms. All items

such as these must be routinely demonstrated to be free from interferences (less than

1/3 the MRL for each method analyte) under the conditions of the analysis by

analyzing LRBs as described in Section 9.3.1. Subtracting blank values from

sample results is not permitted.

4.3 Matrix interferences may be caused by contaminants in the sample. The extent of

matrix interferences will vary considerably from source to source, depending upon the

nature of the water. Humic and/or fulvic material present in the sample at high levels

can cause enhancement and/or suppression in the electrospray ionization source.3-4

Total organic carbon (TOC) is a good indicator of humic content of the sample. Under

the LC conditions used during method development, matrix effects due to total organic

carbon (TOC), up to 8.7 mg/L, were not observed.

4.4 Relatively large quantities of the preservatives (Sect. 8.1.2) are added to sample

bottles. The potential exists for trace-level organic contaminants in these reagents.

Interferences from these sources should be monitored by analysis of LRBs (Sect.

9.3.1), particularly when new lots of reagents are acquired.

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5. SAFETY

The toxicity or carcinogenicity of each reagent used in this method has not been precisely

defined. Each chemical should be treated as a potential health hazard, and exposure to these

chemicals should be minimized. Each laboratory is responsible for maintaining an awareness

of OSHA regulations regarding safe handling of chemicals used in this method. A reference

file of MSDSs should be made available to all personnel involved in the chemical analyses.

Additional references to laboratory safety are available.5-7

6. EQUIPMENT AND SUPPLIES (Brand names and/or catalog numbers are included for

illustration only, and do not imply endorsement of the product.)

6.1 SAMPLE CONTAINERS – Amber glass bottles (40 mL or larger) fitted with teflon-

lined screw caps (Fisher Cat. No.: 02-912-377 or equivalent).

6.2 STANDARD SOLUTION STORAGE CONTAINERS – Amber glass bottles (10 mL

or larger) (Kimble Cat. No.: 60815-1965 or equivalent) fitted with teflon-lined screw

caps (Kimble Cat No.: 73802-15425 or equivalent).

6.3 AUTOSAMPLER VIALS – Amber glass 2.0-mL autosampler vials (National

Scientific Cat. No.: C4000-2W or equivalent) with caps (National Scientific Cat. No.:

C4000-53 or equivalent).

6.4 MICRO SYRINGES – Suggested sizes include 5, 10, 25, 50, 100, 250, 500 and

1000-µL syringes.

6.5 ANALYTICAL BALANCE – Capable of weighing to the nearest 0.0001 g.

6.6 LIQUID CHROMATOGRAPHY (LC)/TANDEM MASS SPECTROMETER

(MS/MS) WITH DATA SYSTEM

6.6.1 LC SYSTEM – Instrument capable of reproducibly injecting at least 50-µL

aliquots, and performing binary linear gradients at a constant flow rate near the

flow rate used for development of this method (0.3 mL/min). The LC must be

capable of pumping the ammonium formate/methanol mobile phase without the

use of a degasser which pulls vacuum on the mobile phase bottle (other types of

degassers are acceptable). Degassers which pull vacuum on the mobile phase

bottle will volatilize the ammonium formate mobile phase causing the analyte

peaks to shift to earlier retention times over the course of the analysis batch. The

use of a column heater is optional.

6.6.2 TANDEM MASS SPECTROMETER – The MS/MS instrument (Waters

Micromass Quattro Premier or equivalent) must be capable of positive ion ESI

near the suggested LC flow rate of 0.3 mL/min. The system must be capable of

performing MS/MS to produce unique product ions (Sect. 3.17) for the method

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analytes within retention time segments. A minimum of 10 scans across the

chromatographic peak is required to ensure adequate precision.

6.6.3 DATA SYSTEM – An interfaced data system is required to acquire, store, reduce,

and output mass spectral data. The computer software should have the capability

of processing stored LC/MS/MS data by recognizing an LC peak within any given

retention time window. The software must allow integration of the ion abundance

of any specific ion within specified time or scan number limits. The software

must be able to construct linear regressions or quadratic calibration curves, and

calculate analyte concentrations.

6.6.4 ANALYTICAL COLUMN – A Waters Atlantis T3 C18 column (2.1 x 150 mm)

packed with 5 µm dp C18 solid phase particles (Cat. No.: 36003736 or equivalent)

was used. Any column that provides adequate resolution, peak shape, capacity,

accuracy, and precision (Sect. 9) may be used.

7. REAGENTS AND STANDARDS

7.1 GASES, REAGENTS, AND SOLVENTS – Reagent grade or better chemicals should

be used. Unless otherwise indicated, it is intended that all reagents shall conform to

the specifications of the Committee on Analytical Reagents of the American Chemical

Society, where such specifications are available. Other grades may be used, provided

it is first determined that the reagent is of sufficiently high purity to permit its use

without lessening the quality of the determination.

7.1.1 REAGENT WATER – Purified water which does not contain any measurable

quantities of any method analytes or interfering compounds greater than 1/3 the

MRL for each method analyte of interest.

7.1.2 METHANOL (CH3OH, CAS#: 67-56-1) – High purity, demonstrated to be free of

analytes and interferences (Fisher Optima LC/MS grade, Cat. No.: A-456 or

equivalent).

7.1.3 AMMONIUM FORMATE (NH4CHO2, CAS#: 540-69-2) – High purity,

demonstrated to be free of analytes and interferences (Sigma-Aldrich LC/MS

grade, Cat. No.: 55674 or equivalent).

7.1.4 AMMONIUM ACETATE (NH4C2H3O2, CAS#: 631-61-8) – High purity,

demonstrated to be free of analytes and interferences (Sigma-Aldrich ACS grade,

Cat. No.: 238074 or equivalent).

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7.1.5 SODIUM OMADINE® (Sodium 2-pyridinethio-1-oxide, CAS#: 3811-73-2) –

High purity demonstrated to be free of analytes and interferences (Sigma-Aldrich

Cat. No.: H3261 or equivalent; Omadine is a registered trademark of Arch

Chemicals, Inc.).

7.1.6 20 mM AMMONIUM FORMATE/REAGENT WATER MOBILE PHASE – To

prepare 1 L, add 1.26 g ammonium formate to 1 L of reagent water. This solution

is prone to volatility losses and should be replaced at least every 48 hours.

7.1.7 2 M AMMONIUM ACETATE/REAGENT WATER – To prepare 100 mL, add

15.4 g ammonium acetate to a 100-mL volumentric flask. Dilute to volume with

reagent water. This solution should be kept refrigerated at ≤6ºC and replaced at

least every two weeks due to potential volatility losses.

7.1.8 32 g/L SODIUM OMADINE® IN REAGENT WATER – To prepare 25 mL, add

0.80 g of sodium omadine to a 25-mL volumetric flask. Dilute to volume with

reagent water. This solution should be kept refrigerated at ≤6ºC.

7.1.9 NITROGEN – Aids in aerosol generation of the ESI liquid spray and is used as

the collision gas in some MS/MS instruments. The nitrogen used should meet or

exceed instrument manufacturer’s specifications.

7.1.10 ARGON – Used as the collision gas in MS/MS instruments. Argon should meet

or exceed instrument manufacturer’s specifications. Nitrogen gas may be used as

the collision gas provided sufficient sensitivity (product ion formation) is

achieved.

7.2 STANDARD SOLUTIONS – When a compound purity is assayed to be 96% or

greater, the weight can be used without correction to calculate the concentration of the

stock standard. Solution concentrations listed in this section were used to develop this

method and are included as an example. Alternate concentrations may be used as

necessary depending on instrument sensitivity and the calibration range used.

Standards for sample fortification generally should be prepared in the smallest volume

that can be accurately measured to minimize the addition of excess organic solvent to

aqueous samples. Even though stability times for standard solutions are

suggested in the following sections, laboratories should use standard QC

practices to determine when their standards need to be replaced.

7.2.1 INTERNAL (IS) STOCK STANDARD SOLUTIONS – This method uses the

five IS compounds listed in the table below. The internal standards were obtained

from Cambridge Isotopes Laboratories, except for methamidophos-d6 which was

obtained from EQ Laboratories. The IS(s) may be purchased from alternate

sources. Although alternate IS standards may be used provided they are

isotopically labeled compounds with similar functional groups as the method

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analytes, the analyst must have documented reasons for using alternate IS(s).

Alternate IS(s) must meet the QC requirements in Section 9.3.4.

Internal Standards

Methamidophos-d6

Acephate-d6

Oxydemeton-methyl-d6

Quinoline-d7

Diisopropyl methylphosphonate-d14 (DIMP-d14)

7.2.1.1 IS STOCK STANDARD SOLUTIONS (100-1000 µg/mL) – These IS stock

standards can be obtained as individual certified stock standard solutions.

During the development of this method, commercially obtained 100 µg/mL

or 1000 µg/mL stock standard solutions in acetonitrile or methanol were

used. IS stock standard solutions were stable for at least six months when

stored at -15 C or less.

7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION (IS PDS) STANDARD

(0.4-12.5 ng/µL) – Prepare, or purchase commercially, the IS PDS at a

suggested concentration of 0.40-12.5 ng/µL in acetonitrile. If prepared from

the individual stock standard solutions (Sect. 7.2.1.1), the table below can be

used as a guideline for preparing the IS PDS. The IS PDS has been shown

to be stable for at least six months when stored in amber glass bottles

(Sect. 6.2) at -4 C. Fortification of the final 1-mL samples with 10 µL of

this 0.40-12.5 ng/µL solution (Sect. 11.2.1) will yield a concentration of

4-125 µg/L of each IS in the 1-mL samples. The total volume of

acetonitrile added to the 1-mL sample should not exceed 2% to avoid

peak broadening of the early eluting analytes. Acetonitrile volumes in

excess of 2% may be used provided the laboratory can document that these

volumes do not adversely affect the peak shape of any of the method

analytes under the laboratories’ operating conditions.

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IS

Conc. of IS

Stock

(µg/mL)

Vol. of IS

Stock

(µL)

Final Conc. of

IS PDS

(ng/µL)a

Methamidophos-d6 100 40 0.40

Acephate-d6 100 40 0.40

Oxydemeton-methyl-d6 100 40 0.40

Quinoline-d7 1000 125 12.5

Diisopropyl methylphosphonate-d14 1000 4.0 0.40

a Final concentrations based upon preparing the IS PDS in a 10-mL volumetric and

diluting to the mark with acetonitrile.

7.2.2 ANALYTE STANDARD SOLUTIONS – Standard solutions may be prepared

from certified, commercially available solutions or from neat compounds. If the

neat compounds used to prepare solutions are of 96% or greater purity, the weight

may be used without correction for purity to calculate the concentration of the

stock standard. Solution concentrations listed in this section were used to

develop this method and are included as example concentrations. With the

exception of dicrotophos and quinoline, the method development work was done

with commercially obtained stock standard solutions, which are readily available

from most suppliers of environmental standards. Quinoline was obtained from

Aldrich and dicrotophos from Chem Service as neat materials. At the time of

method development, DIMP was only available from Cerilliant CIL, Inc. Prepare

the Analyte Stock and Primary Dilution Standards as described below. Analysts

are permitted to use other PDS and calibration standard concentrations and

volumes as necessary to achieve adequate sensitivity.

7.2.2.1 ANALYTE STOCK STANDARD SOLUTION (SSS) (1 mg/mL, except as

noted) – If preparing from neat material, accurately weigh approximately

10 mg of pure material to the nearest 0.1 mg into a tared, 10-mL volumetric

flask. Dilute to the mark with methanol for a final concentration of

1 mg/mL. Repeat for each method analyte. In the case of quinoline,

accurately weigh approximately 20 mg of pure material to the nearest

0.1 mg into a tared, 1-mL volumetric flask. Dilute to the mark with

methanol for a final concentration of 20 mg/mL. These stock standards

were stable for at least six months when stored at -15 C or less.

Alternatively, individual stock standards of the analytes in methanol or

acetonitrile may be purchased commercially. For the development of this

method, commercially purchased stock standards of 1 mg/mL were used to

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make primary dilution standards for all analytes except quinoline and

dicrotophos.

7.2.2.2 METHANOLIC ANALYTE PRIMARY DILUTION STANDARD (MEOH

PDS) SOLUTION (40 -1600 ng/µL) – The analyte MEOH PDS contains all

the method analytes of interest at various concentrations in methanol. The

ESI and MS/MS response varies by compound; therefore, a mix of

concentrations may be needed in the analyte MEOH PDS. During method

development, the analyte MEOH PDS was prepared such that approximately

the same instrument response was obtained for all the analytes. The analyte

MEOH PDS is prepared (table below) by dilution of the combined analyte

SSSs with methanol in a 1-mL volumetric flask and is used to prepare the

analyte WATER PDS (Sect. 7.2.2.3). The analyte MEOH PDS has been

shown to be stable for six months when stored at -15 C. Longer storage

times are acceptable provided appropriate QC measures are documented

demonstrating the analyte MEOH PDS stability.

Analyte

Analyte SSS

Conc.

(mg/mL)

Analyte SSS

Volume

(μL)

Final Analyte

MEOH PDS Conc.

(ng/μL) a

Methamidophos 1.0 40 40

Acephate 1.0 40 40

Aldicarb sulfoxide 1.0 80 80

Oxydemeton-methyl 1.0 40 40

Dicrotophos 1.0 40 40

Aldicarb 1.0 80 80

Quinoline 21.6 80 1728

DIMP 1.0 40 40

Fenamiphos sulfoxide 1.0 40 40

Fenamiphos sulfone 1.0 40 40

Thiofanox 1.0 160 160 a

Final concentration calculation based upon a 1-mL final volume.

7.2.2.3 AQUEOUS ANALYTE PRIMARY DILUTION STANDARD (WATER

PDS) SOLUTION (0.25 -10.7 ng/µL) – The analyte WATER PDS contains

all the method analytes of interest at various concentrations in reagent water

containing 10% methanol. The analyte WATER PDS is prepared by placing

62 µL of the analyte MEOH PDS in a 10-mL volumetric flask and diluting

with reagent water containing 10% methanol and is used to prepare the CAL

standards, and fortify the LFBs, the LFSMs, the LFSMDs and FDs with the

method analytes. The analyte WATER PDS has been shown to be stable for

at least one month when stored at 4 C. Longer storage times are acceptable

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provided appropriate QC measures are documented demonstrating the

analyte WATER PDS stability.

7.2.3 CALIBRATION STANDARDS (CAL) – Prepare a procedural calibration curve

from dilutions of the analyte WATER PDS in preserved reagent water. At least

five to seven calibration concentrations are required to prepare the initial

calibration curve spanning a 50 to 100-fold concentration range (Sect. 10.2).

Prepare the calibration standards, adding appropriate amounts of the ammonium

acetate and sodium omadine concentrated stocks (Sect. 7.1.7 and 7.1.8) as shown

in the table below. The target analyte concentrations found in Tables 5-11 can be

used as a starting point for determining the calibration range. An example of the

dilutions used to prepare the CALs, that were utilized to collect data in Section

17, is shown below. The lowest concentration CAL must be at or below the MRL,

which will depend on system sensitivity. The CALs may also be used as CCCs. If

stored in containers (Sect. 6.2), the aqueous standards must be refrigerated in the

same manner as the samples. A constant amount of the IS PDS is added to each

prepared CAL. This is accomplished for each CAL by taking 990 µL of the final

CAL containing the 20 mM ammonium acetate and 64 mg/L sodium omadine,

and placing it in a 2.0-mL autosampler vial and adding 10 µL of the IS PDS (Sect.

7.2.1.2). During method development, the CAL standards were shown to be

stable for at least two weeks when stored at <6 ºC. Longer storage times are

acceptable provided appropriate QC measures are documented demonstrating the

CAL stability.

CAL

Level

Analyte

WATER

PDS

Conc.

(ng/μL)a

Analyte

WATER

PDS

Volume

(μL)

2 M

Ammonium

Acetate Stock

Volume

(μL)

32 g/L

Sodium

Omadine

Stock Volume

(μL)

Final

CAL

Std.

Conc.

(μg/L) b

Final

Quinoline

Conc.

(μg/L) b

Final

Aldicarb

Conc.

(μg/L) b

Final

Thiofanox

Conc.

(μg/L) b

1 0.25 2 100 20 0.050 2.1 0.10 0.20

2 0.25 5 100 20 0.12 5.4 0.25 0.50

3 0.25 10 100 20 0.25 11 0.50 0.99

4 0.25 20 100 20 0.50 21 0.99 2.0

5 0.25 40 100 20 0.99 43 2.0 4.0

6 0.25 100 100 20 2.5 107 5.0 9.9

7 0.25 200 100 20 5.0 214 10 20 a Quinoline = 10.7 ng/μL, Aldicarb and Aldicarb sulfoxide= 0.50 ng/μL, Thiofanox = 1.0 ng/μL

b Final concentrations based upon preparing the CALs in a 10-mL volumetric and diluting to the mark with reagent

water.

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8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1 SAMPLE BOTTLE PREPARATION

8.1.1 Samples must be collected in amber glass bottles fitted with PTFE-lined screw

caps (Sect. 6.1).

8.1.2 Prior to shipment to the field, ammonium acetate and sodium omadine must be

added to each amber bottle. If using the suggested 40-mL bottles (Sect. 6.1) to

collect a 40-mL aliquot, add 400 μL of the ammonium acetate concentrated stock

(Sect. 7.1.7) and 80 μL of the concentrated sodium omadine stock (Sect. 7.1.8). If

other collection volumes are used, adjust the amount of preservation reagent so

that the final concentrations of ammonium acetate and sodium omadine in the

sample containers are 1.5 g/L and 64 mg/L, respectively. Cap the vials to avoid

evaporation of the preservation reagents.

Compound Amount Purpose

Sodium omadine 64 mg/L Antimicrobial

Ammonium acetate 1.5 g/L Binds free chlorine

8.2 SAMPLE COLLECTION

8.2.1 Open the tap and allow the system to flush until the water temperature has

stabilized (approximately 3 to 5 min). Collect a representative sample from the

flowing stream using a beaker of appropriate size. Use this bulk sample to

generate individual samples as needed. Transfer a volume of approximately

40 mL into each collection container. Alternatively, collect the sample directly in

the sample bottle containing the preservatives.

8.2.2 When filling sample bottles, take care not to flush out the sample preservation

reagents. Samples do not need to be collected headspace free.

8.2.3 After collecting the sample, cap the bottle and agitate by hand to mix the sample

with the preservation reagents. Keep the sample sealed from time of collection

until analysis.

8.3 SAMPLE SHIPMENT AND STORAGE – Samples must be chilled during shipment

and must not exceed 10 °C during the first 48 hours after collection. Sample

temperature must be confirmed to be at or below 10 °C when the samples are received

at the laboratory. Samples stored in the lab must be held at or below 6 °C until

analysis, but should not be frozen.

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NOTE: Samples that are significantly above 10°C, at the time of collection, may

need to be iced or refrigerated for a period of time, in order to chill them

prior to shipping. This will allow them to be shipped with sufficient ice to

meet the above requirements.

NOTE: Samples that arrive at the laboratory on the same day of sample collection

(exclusively due to the close proximity of the sampling site to the

laboratory), may not yet have stabilized to 10ºC or less when they arrive at

the lab. These samples are acceptable ONLY if packed on ice or with frozen

gel packs immediately after sample collection and hence, delivered while

samples are in the process of reaching an equilibrium temperature less than

10ºC. These samples must contain the preservatives as described in Section

8.1.2.

8.4 SAMPLE HOLDING TIMES – Results of the sample storage stability study

(Table 12) indicated that all compounds listed in this method have adequate stability

for 14 days when collected, preserved, shipped and stored as described in Sections 8.1,

8.2, and 8.3. Therefore, aqueous samples must be analyzed within 14 days of

collection.

9. QUALITY CONTROL

9.1 QC requirements include the Initial Demonstration of Capability (IDC) and ongoing

QC requirements that must be met when preparing and analyzing Field Samples. This

section describes the QC parameters, their required frequencies, and the performance

criteria that must be met in order to meet EPA quality objectives. The QC criteria

discussed in the following sections are summarized in Tables 13 and 14. These QC

requirements are considered the minimum acceptable QC criteria. Laboratories are

encouraged to institute additional QC practices to meet their specific needs.

9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) – The IDC must be

successfully performed prior to analyzing any Field Samples. Prior to conducting the

IDC, the analyst must first generate an acceptable Initial Calibration following the

procedure outlined in Section 10.2.

9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND – Any time

a new lot of solvents, reagents, and autosampler vials are used, it must be

demonstrated that an LRB is reasonably free of contamination and that the criteria

in Section 9.3.1 are met.

9.2.2 INITIAL DEMONSTRATION OF PRECISION (IDP) – Prepare and analyze four

to seven replicate LFBs (same as a CCC – Section 9.3.3) fortified near the

midrange of the initial calibration curve according to the procedure described in

Section 11. The sample preservative, as described in Section 8.1.2, must be added

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to these samples. The relative standard deviation (RSD) of the concentrations of

the replicate analyses must be less than 20%.

9.2.3 INITIAL DEMONSTRATION OF ACCURACY (IDA) – Using the same set of

replicate data generated for Section 9.2.2, calculate mean recovery. The mean

recovery of the replicate values must be within ± 30% of the true value.

9.2.4 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION – Establish a target

concentration for the MRL based on the intended use of the method. The MRL

may be established by a laboratory for their specific purpose or may be set by a

regulatory agency. Establish an Initial Calibration following the procedure

outlined in Section 10.2. The lowest CAL standard used to establish the Initial

Calibration (as well as the low-level CCC, Section 10.3) must be at or below the

concentration of the MRL. Establishing the MRL concentration too low may

cause repeated failure of ongoing QC requirements. Confirm the MRL following

the procedure outlined below.

9.2.4.1 Fortify and analyze seven replicate LFBs at the proposed MRL

concentration. These LFBs must contain all method preservatives described

in Section 8.1.2. Calculate the mean measured concentration (Mean) and

standard deviation for the method analytes in these replicates. Determine

the Half Range for the prediction interval of results (HRPIR) for each analyte

using the equation below

HR sPIR 3963.

where

s = the standard deviation

3.963 = a constant value for seven replicates.1

9.2.4.2 Confirm that the upper and lower limits for the Prediction Interval of Result

(PIR = Mean + HRPIR) meet the upper and lower recovery limits as shown

below

The Upper PIR Limit must be ≤150% recovery.

150% %100ononcentratiFortifiedC

HRMean PIR

The Lower PIR Limit must be 50% recovery.

50% %100ononcentratiFortifiedC

HRMean PIR

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9.2.4.3 The MRL is validated if both the Upper and Lower PIR Limits meet the

criteria described above (Sect. 9.2.4.2). If these criteria are not met, the

MRL has been set too low and must be confirmed again at a higher

concentration.

9.2.5 CALIBRATION CONFIRMATION – Analyze a QCS as described in

Section 9.3.7 to confirm the accuracy of the standards/calibration curve.

9.2.6 DETECTION LIMIT DETERMINATION (optional) – While DL determination

is not a specific requirement of this method, it may be required by various

regulatory bodies associated with compliance monitoring. It is the responsibility

of the laboratory to determine if DL determination is required based upon the

intended use of the data.

Replicate analyses for this procedure should be done over at least three days (i.e.,

both the sample preparation and the LC/MS/MS analyses should be done over at

least three days). Prepare at least seven replicate LFBs at a concentration

estimated to be near the DL (e.g., three LFBs individually fortified on day one,

two LFBs individually fortified on day two, and two LFBs individually fortified

on day three). This concentration may be estimated by selecting a concentration

at two to five times the noise level. The DLs in Table 5 were calculated from

LFBs fortified at various concentrations as indicated in the table. The

appropriate fortification concentrations will be dependent upon the sensitivity of

the LC/MS/MS system used. All preservation reagents listed in Section 8.1.2

must also be added to these samples. Analyze the seven replicates through all

steps of Section 11.

NOTE: If an MRL confirmation data set meets these requirements, a DL may be

calculated from the MRL confirmation data, and no additional analyses

are necessary.

Calculate the DL using the following equation

)99.01 ,1(n

tsDL

where

s = standard deviation of replicate analyses

t (n-1, 1-α=0.99) = Student's t value for the 99% confidence

level with n-1 degrees of freedom

n = number of replicates.

NOTE: Do not subtract blank values when performing DL calculations. The DL

is a statistical determination of precision only.2 If the DL replicates are

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fortified at a low enough concentration, it is likely that they will not meet

the precision and accuracy criteria for CCCs, and may result in a

calculated DL that is higher than the fortified concentration. Therefore,

no precision and accuracy criteria are specified.

9.3 ONGOING QC REQUIREMENTS – This section summarizes the ongoing QC

criteria that must be followed when processing and analyzing Field Samples.

9.3.1 LABORATORY REAGENT BLANK (LRB) – An LRB is required with each

analysis batch (Sect. 3.1) to confirm that potential background contaminants are

not interfering with the identification or quantitation of method analytes. If the

LRB produces a peak within the retention time window of any analyte that would

prevent the determination of that analyte, determine the source of contamination

and eliminate the interference before processing samples. Background

contamination must be reduced to an acceptable level before proceeding.

Background from method analytes or other contaminants that interfere with the

measurement of method analytes must be below 1/3 of the MRL. Blank

contamination is estimated by extrapolation, if the concentration is below the

lowest CAL standard. This extrapolation procedure is not allowed for sample

results as it may not meet data quality objectives. If the method analytes are

detected in the LRB at concentrations equal to or greater than 1/3 the MRL, then

all data for the problem analyte(s) must be considered invalid for all samples in

the analysis batch.

9.3.2 CONTINUING CALIBRATION CHECK (CCC) – CCC standards, containing

the preservatives, are analyzed at the beginning of each analysis batch, after every

10 Field Samples, and at the end of the analysis batch. See Section 10.3 for

concentration requirements and acceptance criteria.

9.3.3 LABORATORY FORTIFIED BLANK (LFB) – Since this method utilizes

procedural calibration standards, which are fortified reagent waters, there is no

difference between the LFB and the CCC. Consequently, the analysis of a

separate LFB is not required as part of the ongoing QC. However, the acronym

LFB is used for clarity in the IDC.

9.3.4 INTERNAL STANDARDS (IS) – The analyst must monitor the peak areas of the

IS(s) in all injections during each analysis day. The IS peak areas in any

chromatographic run must be within 50-150% of the average IS areas in the most

recent calibration curve. If the IS areas in a chromatographic run do not meet

these criteria, inject a second aliquot from the same autosampler vial.

9.3.4.1 If the reinjected aliquot produces an acceptable IS response, report results

for that aliquot.

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9.3.4.2 If the reinjected aliquot fails the IS criterion, the analyst should check the

calibration by reanalyzing the most recently acceptable CAL standard. If

the CAL standard fails the criteria of Section 10.3, recalibration is in order

per Section 10.2. If the CAL standard is acceptable, report results obtained

from the reinjected aliquot, but annotate as “suspect/IS recovery.”

Alternatively, prepare another aliquot of the sample as specified in Section

11.2 or collect a new sample and re-analyze.

9.3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – Analysis of an

LFSM is required in each analysis batch and is used to determine that the sample

matrix does not adversely affect method accuracy. Assessment of method

precision is accomplished by analysis of a Field Duplicate (FD) (Sect. 9.3.6);

however, infrequent occurrence of method analytes would hinder this assessment.

If the occurrence of method analytes in the samples is infrequent, or if historical

trends are unavailable, a second LFSM, or LFSMD, must be prepared and

analyzed from a duplicate of the Field Sample. Analysis batches that contain

LFSMDs will not require the analysis of a FD. If a variety of different sample

matrices are analyzed regularly, for example, drinking water from groundwater

and surface water sources, method performance should be established for each.

Over time, LFSM data should be documented by the laboratory for all routine

sample sources.

9.3.5.1 Within each analysis batch (Sect. 3.1), a minimum of one Field Sample is

fortified as an LFSM for every 20 Field Samples analyzed. The LFSM is

prepared by spiking a sample with an appropriate amount of the Analyte

WATER PDS (Sect. 7.2.2.3). Select a spiking concentration that is greater

than or equal to the matrix background concentration, if known. Use

historical data and rotate through the low, mid and high concentrations when

selecting a fortifying concentration.

9.3.5.2 Calculate the percent recovery (%R) for each analyte using the equation

where

A = measured concentration in the fortified sample

B = measured concentration in the unfortified sample

C = fortification concentration.

9.3.5.3 Analyte recoveries may exhibit matrix bias. For samples fortified at or

above their native concentration, recoveries should range between 70-130%,

except for low-level fortification near or at the MRL (within a factor of

two-times the MRL concentration) where 50-150% recoveries are

acceptable.

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9.3.5.4 If the accuracy of any analyte falls outside the designated range in the

LFSM, and the laboratory performance for that analyte is shown to be in

control in the CCCs, and the CCCs for the batch were not freshly prepared

on the day of analysis, the Analyte WATER PDS used to fortify the matrix

sample must be checked for analyte losses. Check the accuracy of the

Analyte WATER PDS by using it to prepare a fresh CCC. The fresh CCC

must meet the criteria in Section 10.3. If the fresh CCC does not meet the

criteria in Section 10.3, a new Analyte WATER PDS must be prepared and

the LFMS analysis repeated.

9.3.5.5 If the accuracy of any analyte falls outside the designated range, and the

laboratory performance for that analyte is shown to be in control in the

CCCs, as well as in the Analyte WATER PDS, the recovery is judged to be

matrix biased. The result for that analyte in the unfortified sample is labeled

“suspect/matrix” to inform the data user that the results are suspect due to

matrix effects.

9.3.6 FIELD DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX

DUPLICATE (FD or LFSMD) – Within each analysis batch (not to exceed 20

Field Samples, Sect. 3.1), a minimum of one FD or LFSMD must be analyzed.

Duplicates check the precision associated with sample collection, preservation,

storage, and laboratory procedures. If method analytes are not routinely observed

in Field Samples, an LFSMD should be analyzed rather than an FD.

9.3.6.1 Calculate the relative percent difference (RPD) for duplicate measurements

(FD1 and FD2) using the equation

1002/21

21

FDFD

FDFDRPD

9.3.6.2 RPDs for FDs should be ≤30%. Greater variability may be observed when

FDs have analyte concentrations that are within a factor of two of the MRL.

At these concentrations, FDs should have RPDs that are ≤50%. If the RPD

of any analyte falls outside the designated range, and the laboratory

performance for that analyte is shown to be in control in the CCC, the

recovery is judged to be matrix biased. The result for that analyte in the

unfortified sample is labeled “suspect/matrix” to inform the data user that

the results are suspect due to matrix effects.

9.3.6.3 If an LFSMD is analyzed instead of a FD, calculate the RPD for duplicate

LFSMs (LFSM and LFSMD) using the equation

538-21

1002/LFSMDLFSM

LFSMDLFSMRPD

9.3.6.4 RPDs for duplicate LFSMs should be ≤30% for samples fortified at or

above their native concentration. Greater variability may be observed when

LFSMs are fortified at analyte concentrations that are within a factor of two

of the MRL. LFSMs fortified at these concentrations should have RPDs

that are ≤50%. If the RPD of any analyte falls outside the designated range,

and the laboratory performance for that analyte is shown to be in control in

the CCC, the recovery is judged to be matrix biased. The result for that

analyte in the unfortified sample is labeled “suspect/matrix” to inform the

data user that the results are suspect due to matrix effects.

9.3.7 QUALITY CONTROL SAMPLES (QCS) – As part of the IDC (Sect. 9.2), each

time a new Analyte MEOH PDS (Sect. 7.2.2.2) is prepared, and at least quarterly,

analyze a QCS sample from a source different from the source of the CAL

standards. If a second vendor is not available, then a different lot of the standard

should be used. The QCS should be prepared near the midpoint of the calibration

range and analyzed as a CCC. Acceptance criteria for the QCS are identical to the

CCCs; the calculated amount for each analyte must be ± 30% of the expected

value. If measured analyte concentrations are not of acceptable accuracy, check

the entire analytical procedure to locate and correct the problem.

9.4 METHOD MODIFICATION QC REQUIREMENTS – The analyst is permitted to

modify LC columns, LC conditions, internal standards, and MS and MS/MS

conditions. Each time such method modifications are made, the laboratory must

confirm that the modifications provide acceptable method performance as defined in

the Sections 9.4.1-9.4.3. Modifications to LC conditions should still produce

conditions such that co-elution of the method analytes is minimized to reduce the

probability of ESI suppression/enhancement effects.

9.4.1 Each time method modifications are made, the analyst must repeat the procedures

of the IDC (Sect. 9.2) and verify that all QC criteria can be met in ongoing QC

samples (Sect. 9.3).

9.4.2 The analyst is also required to evaluate and document method performance for the

proposed method modifications in real matrices that span the range of waters that

the laboratory analyzes. This additional step is required because modifications

that perform acceptably in the IDC, which is conducted in reagent water, can fail

ongoing method QC requirements in real matrices. This is particularly important

for methods subject to matrix effects. If, for example, the laboratory analyzes

finished waters from both surface and groundwater municipalities, this

requirement can be accomplished by assessing precision and accuracy (Sects.

9.2.2 and 9.2.3) in a surface water with moderate to high Total Organic Carbon

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(TOC) (e.g., 2 mg/L or greater) and a hard groundwater (e.g., 250 mg/L or greater

as calcium carbonate).

9.4.3 The results of Sections 9.4.1 and 9.4.2 must be appropriately documented by the

analyst and should be independently assessed by the laboratory’s Quality

Assurance (QA) officer prior to analyzing Field Samples.

9.4.3.1 When implementing method modifications, it is the responsibility of the

laboratory to closely review the results of ongoing QC, and in particular,

the results associated with the LFSMs (Sect. 9.3.5), FDs or LFSMDs

(Sect. 9.3.6), CCCs (Sect. 9.3.2), and the IS area counts (Sect. 9.3.4). If

repeated failures are noted, the modification must be abandoned.

10. CALIBRATION AND STANDARDIZATION

10.1 Demonstration and documentation of acceptable initial calibration is required before

any samples are analyzed. After the initial calibration is successful, a CCC is required

at the beginning and end of each period in which analyses are performed, and after

every tenth Field Sample.

10.2 INITIAL CALIBRATION

10.2.1 ESI-MS/MS TUNE

10.2.1.1 Calibrate the mass scale of the MS with the calibration compounds and

procedures prescribed by the manufacturer.

10.2.1.2 Optimize the [M+H]+ for each method analyte by infusing approximately

1.0-5.0 µg/mL of each analyte (prepared in water containing 10% methanol)

directly into the MS at the chosen LC mobile phase flow rate

(approximately 0.3 mL/min). This tune can be done on a mix of the method

analytes. The MS parameters (voltages, temperatures, gas flows, etc.) are

varied until optimal analyte responses are determined. The method analytes

may have different optima requiring some compromise between the optima.

See Table 2 for ESI-MS conditions used in method development.

10.2.1.3 Optimize the product ion (Sect. 3.17) for each analyte by infusing

approximately 1.0-5.0 µg/mL of each analyte (prepared in the initial mobile

phase conditions) directly into the MS at the chosen LC mobile phase flow

rate (approximately 0.3 mL/min). This tune can be done on a mix of the

method analytes. The MS/MS parameters (collision gas pressure, collision

energy, etc.) are varied until optimal analyte responses are determined. See

Table 4 for MS/MS conditions used in method development.

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10.2.2 Establish LC operating parameters that optimize resolution and peak shape.

Suggested LC conditions can be found in Table 1. The LC conditions listed in

Table 1 may not be optimum for all LC systems and may need to be optimized by

the analyst. If possible, optimize chromatographic conditions such that a unique

quantitation ion is available for each analyte that is free from interference due to

an identical product ion in any co-eluting (or overlapping) peak(s).

NOTE: During method development, the LC flow was diverted to waste for the

first three minutes of the analysis. The flow was diverted to prevent

potential fouling of the ESI source from sample components including

preservatives. Laboratories are not required to divert the LC flow, but

more frequent maintenance may be necessary if the flow is not diverted

prior to elution of the first analyte.

10.2.3 Inject a mid-level CAL standard under LC/MS conditions to obtain the retention

times of each method analyte. Divide the chromatogram into retention time

windows so that each window contains one or more chromatographic peaks.

During MS/MS analysis, fragment a small number of selected precursor ions

([M+H]+; Sect. 3.15) for the analytes in each window and choose the most

abundant product ion. The product ions (also the quantitation ions) chosen during

method development are in Table 4, although these will be instrument dependent.

For maximum sensitivity during method development, small mass windows of

±0.5 daltons around the product ion mass were used for quantitation.

10.2.4 Inject a mid-level CAL standard under optimized LC/MS/MS conditions to ensure

that each method analyte is observed in its MS/MS window and that there are at

least 10 scans across the peak for optimum precision.

CAUTION: When acquiring MS/MS data, LC operating conditions must be

carefully reproduced for each analysis to provide reproducible

retention times. If this is not done, the correct ions will not be

monitored at the appropriate times. As a precautionary measure,

the chromatographic peaks in each window must not elute too close

to the edge of the segment time window.

10.2.5 Prepare a set of at least five CAL standards as described in Section 7.2.3. The

lowest concentration CAL standard must be at or below the MRL, which will

depend on system sensitivity. It is recommended that at least four of the CAL

standards are at a concentration greater than or equal to the MRL.

10.2.6 The LC/MS/MS system is calibrated using the IS technique. Use the LC/MS/MS

data system software to generate a linear regression or quadratic calibration curve

for each of the analytes. The curves may be concentration weighted, if necessary.

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10.2.7 CALIBRATION ACCEPTANCE CRITERIA – When quantitated using the initial

calibration curve, each calibration point, except the lowest point, for each analyte

should calculate to be within 70-130% of its true value. The lowest CAL point

should calculate to be within 50-150% of its true value. If these criteria cannot be

met, the analyst will have difficulty meeting ongoing QC criteria. It is

recommended that corrective action is taken to reanalyze the CAL standards,

restrict the range of calibration, or select an alternate method of calibration.

10.3 CONTINUING CALIBRATION CHECK (CCC) – Minimum daily calibration

verification is as follows. Verify the initial calibration at the beginning and end of

each group of analyses, and after every tenth sample during analyses. In this context,

a “sample” is considered to be a Field Sample. LRBs, CCCs, LFSMs, FDs and

LFSMDs are not counted as samples. The beginning CCC of each analysis batch must

be at or below the MRL in order to verify instrument sensitivity prior to any analyses.

If standards have been prepared such that all low CAL points are not in the same CAL

solution, it may be necessary to analyze two CAL standards to meet this requirement.

Alternatively, the analyte concentrations in the analyte PDS may be customized to

meet this criteria. Subsequent CCCs should alternate between a medium and high

concentration CAL standard.

10.3.1 Inject an aliquot of the appropriate concentration CAL standard and analyze with

the same conditions used during the initial calibration.

10.3.2 Determine that the absolute areas of the quantitation ions of the IS(s) are within

50-150% of the average areas measured in the most recent calibration. If any of

the IS areas has changed by more than these amounts, adjustments must be made

to restore system sensitivity. These adjustments may include cleaning of the MS

ion source, or other maintenance as indicated in Section 10.3.4. Major

instrument maintenance requires recalibration (Sect 10.2) and verification of

sensitivity by analyzing a CCC at or below the MRL (Sect 10.3). Control charts

are useful aids in documenting system sensitivity changes.

10.3.3 Calculate the concentration of each analyte in the CCC. The calculated amount

for each analyte for medium and high level CCCs must be within ± 30% of the

true value. The calculated amount for the lowest calibration point for each

analyte must be within ± 50% of the true value. If these conditions cannot be met,

then all data for the problem analyte must be considered invalid, and remedial

action should be taken (Sect. 10.3.4). This may require recalibration. Any Field

or QC Samples that have been analyzed since the last acceptable calibration

verification should be reanalyzed after adequate calibration has been restored,

with the following exception. If the CCC at the end of the batch fails because

the calculated concentration is greater than 130% (150% for the low-level

CCC) for a particular method analyte, and Field Samples show no detection

for that method analyte, non-detects may be reported without re-analysis.

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10.3.4 REMEDIAL ACTION – Failure to meet CCC QC performance criteria may

require remedial action. Major maintenance, such as cleaning the electrospray

probe, atmospheric pressure ionization source, cleaning the mass analyzer,

replacing the LC column, etc., requires recalibration (Sect 10.2) and verification

of sensitivity by analyzing a CCC at or below the MRL (Sect 10.3).

11. PROCEDURE

11.1. SAMPLE PREPARATION

11.1.1. Samples are preserved, collected and stored as presented in Section 8. All Field

and QC Samples must contain the preservatives listed in Section 8.1.2, including

the LRB.

11.1.2. In addition to the preservatives, if the sample is an LFSM or LFSMD, add the

necessary amount of analyte WATER PDS (Sect. 7.2.2.3). Cap and invert each

sample to mix.

NOTE: If the laboratory is concerned that a particular matrix may contain high

particulate levels that could cause clogging of the LC system, sample

filtration may be incorporated into the procedure. If filtering is

incorporated as part of the sample preparation, the first lot of filters must

be subjected to the procedures outlined in the IDC (Sect. 9.2) and meet

the acceptance criteria defined in Section 9.2 to ensure that they do not

introduce interferences or retain any of the method analytes. Verification

of subsequent lots of filters can be accomplished by examining a filtered

LRB and duplicate samples of filtered LFBs fortified at the MRL. The

filtered LFBs should calculate to be within 50% of the true value. If the

LRB or the LFBs fail this evaluation, the full IDC will need to be

repeated with the new lot of filters. CAL standards and CCCs should not

be filtered in order to identify potential losses associated with the sample

filtration devices. During method development, Pall Gelman GHP

Acrodisc, 25 mm syringe filters with 0.45 µm GHP membranes (Cat.

No.: 4560T) were evaluated and found to pass all QC criteria. Other

filter materials may be used provided the QC criteria in Section 9 are

met.

11.2. SAMPLE ANALYSIS

11.2.1. Transfer a 990 µL aliquot of the sample to an autosampler vial. Add 10 µL of the

IS PDS (Sect. 7.2.1.2) to the autosampler vial. Cap and invert each vial to mix.

11.2.2. Establish operating conditions equivalent to those summarized in Tables 1-4 of

Section 17. Instrument conditions and columns should be optimized prior to the

initiation of the IDC.

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11.2.3. Establish an appropriate retention time window for each analyte. This should be

based on measurements of actual retention time variation for each method analyte

in CAL standard solutions analyzed on the LC over the course of time. A value

of plus or minus three times the standard deviation of the retention time, obtained

for each method analyte while establishing the initial calibration and completing

the IDC, can be used to calculate a suggested window size. However, the

experience of the analyst should weigh heavily on the determination of the

appropriate retention window size.

11.2.4. Calibrate the system by either the analysis of a set of CAL standards (Sect. 10.2)

or by confirming the existing calibration is still valid by analyzing a CCC as

described in Section 10.3. If establishing an initial calibration, complete the IDC

as described in Section 9.2.

11.2.5. Begin analyzing Field Samples, including QC samples, at their appropriate

frequency by injecting the same size aliquots (50 µL was used in method

development) under the same conditions used to analyze the CAL standards.

11.2.6. At the conclusion of data acquisition, use the same software that was used in the

calibration procedure to identify peaks of interest in predetermined retention time

windows. Use the data system software to examine the ion abundances of the

peaks in the chromatogram. Identify an analyte by comparison of its retention

time with that of the corresponding method analyte peak in a reference standard.

Comparison of the MS/MS mass spectra is not particularly useful given the

limited ±0.5 dalton mass range around a single product ion for each method

analyte.

11.2.7. The analyst must not extrapolate beyond the established calibration range. If an

analyte peak area exceeds the range of the calibration curve, the new aliquot of

sample may be diluted with reagent water and the appropriate amount of IS

added. Re-inject the diluted sample. Incorporate the dilution factor into the final

concentration calculations. The resulting data should be documented as a

dilution, with an increased MRL.

12. DATA ANALYSIS AND CALCULATION

12.1. In validating this method, concentrations were calculated by measuring the product

ions listed in Table 4. Other product ions may be selected at the discretion of the

analyst.

12.2. Calculate analyte concentrations using the multipoint calibration established in

Section 10.2. Do not use daily calibration verification data to quantitate analytes in

samples.

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12.3 Prior to reporting the data, the chromatogram should be reviewed for any incorrect

peak identification or poor integration.

12.4 Calculations must utilize all available digits of precision, but final reported

concentrations should be rounded to an appropriate number of significant figures (one

digit of uncertainty), typically two, and not more than three significant figures.

13. METHOD PERFORMANCE

13.1 PRECISION, ACCURACY, AND MINIMUM REPORTING LEVELS – Tables for

these data are presented in Section 17. LCMRLs and DLs for each method analyte are

presented in Table 5. Precision and accuracy are presented for four tap water matrices:

reagent water (Tables 6 and 7); chlorinated surface water (Tables 8 and 9); chlorinated

(finished) ground water (Table 10); chlorinated surface water fortified with natural

organic matter (Table 11).

13.2 SAMPLE STORAGE STABILITY STUDIES – An analyte storage stability study was

conducted by fortifying the analytes into chlorinated surface water samples that were

collected, preserved, and stored as described in Section 8. The chlorinated surface

water was adjusted to pH=9.0 before adding the preservatives and analytes to simulate

the worst case scenario for compounds with potential to degrade under basic pH

conditions. The precision and mean recovery of analyses (n=7), conducted on Days 0,

7, and 14, are presented in Table 12.

13.3 MULTIPLE LABORATORY VERIFICATION – The performance of this method

was demonstrated by multiple laboratories, with accuracy and precision results similar

to those reported in Section 17. The authors wish to acknowledge the assistance of the

analysts and laboratories listed below for their participation in the multi-lab

demonstration.

13.3.1 Dr. Andrew Eaton and Mr. Ali Haghani of MWH Laboratories, Monrovia, CA.

13.3.2 Dr. Yongtao Li of Underwriters Laboratories, Inc., South Bend, IN.

13.3.3 Ms. Tiffany Payne and Mr. Ed George of Varian, Inc., Walnut Creek, CA.

14. POLLUTION PREVENTION

14.1 This method utilizes DAI-LC/MS/MS for the analysis of method analytes in water. It

requires the use of very small volumes of organic solvent and very small quantities of

pure analytes, thereby minimizing the potential hazards to both the analyst and the

environment.

14.2 For information about pollution prevention that may be applicable to laboratory

operations, consult “Less is Better: Laboratory Chemical Management for Waste

538-28

Reduction” available from the American Chemical Society’s Department of

Government Relations and Science Policy, 1155 16th

Street N.W., Washington, D.C.,

20036 or on-line at http://membership.acs.org/c/ccs/pub_9.htm (accessed November

2009).

15. WASTE MANAGEMENT

15.1 The analytical procedures described in this method generate relatively small amounts of

waste since only small amounts of reagents and solvents are used. The matrices of

concern are finished drinking water or source water. However, laboratory waste

management practices must be conducted consistent with all applicable rules and

regulations, and that laboratories protect the air, water, and land by minimizing and

controlling all releases from fume hoods and bench operations. Also, compliance is

required with any sewage discharge permits and regulations, particularly the hazardous

waste identification rules and land disposal restrictions.

15.2 Local regulations may prohibit sink disposal of water containing sodium omadine.

Therefore, the laboratory should determine with local officials how to safely dispose of

Field and QC samples containing sodium omadine.

16. REFERENCES

1. Winslow, S.D., Pepich, B.V., Martin, J.J., Hallberg, G.R., Munch, D.J., Frebis, C.P.,

Hedrick, E.J., Krop, R.A. “Statistical Procedures for Determination and Verification of

Minimum Reporting Levels for Drinking water Methods.” Environ. Sci. & Technol. 2004,

40, 281-288.

2. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, W.L. Budde, "Trace Analyses for

Wastewaters.” Environ. Sci. Technol. 1981, 15, 1426-1435.

3. Leenheer, J.A., Rostad, C.E., Gates, P.M., Furlong, E.T., Ferrer, I. “Molecular Resolution

and Fragmentation of Fulvic Acid by Electrospray Ionization/Multistage Tandem Mass

Spectrometry.” Anal. Chem. 2001, 73, 1461-1471.

4. Cahill, J.D., Furlong E.T., Burkhardt, M.R., Kolpin, D., Anderson, L.G. “Determination of

Pharmaceutical Compounds in Surface- and Ground-Water Samples by Solid-Phase

Extraction and High-Performance Liquid Chromatography Electrospray Ionization Mass

Spectrometry.” J. Chromatogr. A 2004, 1041, 171-180.

5. “OSHA Safety and Health Standards, General Industry," (29CRF1910). Occupational Safety

and Health Administration, OSHA 2206, (Revised, July 1, 2001).

6. “Carcinogens-Working with Carcinogens,” Publication No. 77-206, Department of Health,

Education, and Welfare, Public Health Service, Center for Disease Control, National Institute

of Occupational Safety and Health, Atlanta, Georgia, August 1977.

538-29

7. "Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,

Committee on Chemical Safety, 7th Edition. Information on obtaining a copy is available at

http://membership.acs.org/C/CCS/pub_3.htm (accessed November, 2009). Also available by

request at [email protected].

http://membership.acs.org/C/CCS/pub_3.htm

mailto:[email protected]

538-30

17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA

TABLE 1. LC METHOD CONDITIONS

Time (min) % 20 mM Ammonium Formate % Methanol

Initial 90.0 10.0

3.0 90.0 10.0

5.0 70.0 30.0

8.0 70.0 30.0

20 30.0 70.0

20.1 10.0 90.0

25 10.0 90.0

25.1 90.0 10.0

30 90.0 10.0

Waters Atlantis T3 2.1 x 150 mm packed with 5.0 µm C18 stationary phase

Flow rate of 0.3 mL/min

50 µL injection

TABLE 2. ESI-MS METHOD CONDITIONS

ESI Conditions

Polarity Positive ion

Capillary needle voltage +4 kV

Cone gas flow 100 L/hr

Nitrogen desolvation gas flow 1000 L/hr

Desolvation gas temp. 350ºC

538-31

TABLE 3. METHOD ANALYTE RETENTION TIMES (RTs), AND

SUGGESTED IS REFERENCES

Analyte Peak #

(Fig. 1)

RT

(min)

IS#

Ref

Methamidophos 2 3.66 1

Acephate 4 5.24 2

Aldicarb sulfoxide 5 6.51 2

Oxydemeton-methyl 7 7.50 3

Dicrotophos 8 9.59 3

Aldicarb 9 14.74 3

Quinoline 11 15.77 4

DIMP 13 16.65 5

Fenamiphos sulfoxide 14 17.63 3

Fenamiphos sulfone 15 18.07 3

Thiofanox 16 18.79 5

Methamidophos-d6 1 3.57 IS#1

Acephate-d6 3 5.16 IS#2

Oxydemeton-methyl-d6 6 7.44 IS#3

Quinoline-d7 10 15.57 IS#4

DIMP-d14 12 16.44 IS#5

538-32

TABLE 4. MS/MS METHOD CONDITIONSa,b

Segmentc Analyte

Precursor Iond

(m/z)

Product Iond,e

(m/z)

Cone

Voltage

(v)

Collision

Energyf

(v)

1 Methamidophos 142 94 20 15

1 Acephate 184 143 20 15

2 Aldicarb sulfoxide 207 132 20 10

2 Oxydemeton-methyl 247 169 20 15

3 Dicrotophos 238 112 25 15

3 Aldicarb 208 89 10 15

4 Quinoline 130 77 35 30

4 DIMP 181 97 15 10

4 Fenamiphos sulfoxide 320 233 30 25

4 Fenamiphos sulfone 336 266 30 20

5 Thiofanox 219 57 20 20

1 Methamidophos-d6 148 97 20 15

1 Acephate-d6 190 149 20 15

2 Oxydemeton-methyl-d6 253 175 20 15

4 Quinoline-d7 137 81 35 30

4 DIMP-d14 195 99 15 15 a An LC/MS/MS chromatogram of the analytes is shown in Figure 1.

b These conditions were optimized during method development and used to collect the data in

Section 17. Optimum conditions may vary on different LC/MS/MS instruments. c

Segments are time durations in which single or multiple scan events occur. d Precursor and product ions listed in this table are nominal masses. During MS and MS/MS

optimization, the analyst should determine the precursor and product ion masses to one

decimal place by locating the apex of the mass spectral peak (e.g., m/z 141.9 93.9 for

methamidophos). These precursor and product ion masses (with one decimal place) should be

used in the MS/MS method for all analyses. e Ions used for quantitation purposes.

f Argon used as collision gas at a flow rate of 0.3 mL/min.

538-33

TABLE 5. DLs AND LCMRLs IN REAGENT WATER

Analyte

Fortified Conc.

(µg/L)a

DLb

(µg/L)

LCMRLc

(µg/L)

Methamidophos 0.050 0.017 0.032

Acephate 0.050 0.019 0.044

Aldicarb sulfoxide 0.10 0.060 0.088

Oxydemeton-methyl 0.050 0.010 0.019

Dicrotophos 0.050 0.025 0.039

Aldicarb 0.10 0.030 0.030

Quinoline 2.1 1.2 1.5

DIMP 0.050 0.014 0.022

Fenamiphos sulfoxide 0.050 0.034 0.042

Fenamiphos sulfone 0.050 0.0087 0.011

Thiofanox 0.20 0.090 0.18 a Spiking concentration used to determine DL.

b Detection limits were determined by analyzing eight replicates over three days according to

Section 9.2.6. c LCMRLs were calculated according to the procedure in reference 1.

TABLE 6. PRECISION AND ACCURACY IN REAGENT WATER FORTIFIED AT

0.05-2.1 µg/L (n=8)

Analyte Fortified Conc. (µg/L) Mean % Recovery % RSD


Acephate 0.050 96.0 13

Aldicarb sulfoxide 0.10 96.9 21

Oxydemeton-methyl 0.050 105 6.5

Dicrotophos 0.050 114 15

Aldicarb 0.10 106 9.5

Quinoline 2.1 94.0 20

DIMP 0.050 103 9.2


Fenamiphos sulfone 0.050 108 5.4

Thiofanox 0.20 112 13

538-34

TABLE 7. PRECISION AND ACCURACY IN REAGENT WATER FORTIFIED AT

0.99-43 µg/L (n=7)


Methamidophos 0.99 105 1.3

Acephate 0.99 107 4.3

Aldicarb sulfoxide 2.0 105 1.9


Dicrotophos 0.99 95.9 5.2

Aldicarb 2.0 97.1 4.9

Quinoline 43 101 5.3

DIMP 0.99 101 3.3

Fenamiphos sulfoxide 0.99 94.5 6.5

Fenamiphos sulfone 0.99 92.3 4.7

Thiofanox 4.0 93.2 4.2

TABLE 8. PRECISION AND ACCURACY IN CHLORINATED SURFACE WATER

FORTIFIED AT 0.05-2.1 µg/L (n=7)



Acephate 0.050 113 18

Aldicarb sulfoxide 0.10 106 18

Oxydemeton-methyl 0.050 99.8 14

Dicrotophos 0.050 93.9 16

Aldicarb 0.10 116 15

Quinoline 2.1 97.4 19

DIMP 0.050 98.9 15


Fenamiphos sulfone 0.050 111 18

Thiofanox 0.20 119 14

538-35


FORTIFIED AT 0.99-43 µg/La (n=7)



Acephate 0.99 103 3.2




Aldicarb 2.0 112 2.1

Quinoline 43 98.5 3.7

DIMP 0.99 102 2.7

Fenamiphos sulfoxide 0.99 110 3.5


Thiofanox 4.0 95.2 3.3 a TOC = 1.49 mg/L and hardness = 120 mg/L as calcium carbonate.

TABLE 10. PRECISION AND ACCURACY IN FORTIFIED CHLORINATED

GROUND WATER FORTIFIED AT 0.99-43 µg/La (n=7)


Methamidophos 0.99 98.1 7.0

Acephate 0.99 106 4.4

Aldicarb sulfoxide 2.0 102 5.8





DIMP 0.99 99.2 4.5



Thiofanox 4.0 89.3 2.4 a TOC = 0.726 mg/L and hardness = 342 mg/L as calcium carbonate.

538-36


CONTAINING NATURAL ORGANIC MATERIALa AND FORTIFIED

WITH ANALYTES AT 0.99-43 µg/L (n=5)



Acephate 0.99 106 3.8



Dicrotophos 0.99 102 3.4



DIMP 0.99 103 1.8



Thiofanox 4.0 104 4.9 a Prepared using commercially available (International Humic Substances Society) aquatic

natural organic matter (NOM) isolated from the Suwannee River. The chlorinated surface

water was fortified with NOM to obtain a TOC measurement of 8.7 mg/L.

538-37

TABLE 12. AQUEOUS SAMPLE HOLDING TIME DATA FOR SAMPLES FROM CHLORINATED SURFACE WATERa,

FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STORED ACCORDING TO SECTION 8

(n=7)

Analyte

Fortified

Conc.

(µg/L)

Day 0 Day 7 Day 14

Mean %Rec % RSD Mean %Rec % RSD Mean %Rec % RSD

Methamidophos 0.99 94.4 2.8 97.5 1.9 91.1 2.5

Acephate 0.99 105 3.3 106 4.4 94.7 4.1

Aldicarb sulfoxide 2.0 97.7 2.9 100 6.2 84.5 3.2

Oxydemeton-methyl 0.99 92.9 3.4 85.9 2.5 80.0 2.3

Dicrotophos 0.99 91.4 5.7 94.5 2.9 93.1 4.6

Aldicarb 2.0 104 4.4 110 3.7 97.6 2.6

Quinoline 43 97.8 2.6 98.0 4.0 99.9 2.4

DIMP 0.99 96.8 2.7 98.5 3.6 98.3 1.2

Fenamiphos sulfoxide 0.99 102 4.4 112 3.3 99.0 3.0

Fenamiphos sulfone 0.99 101 4.3 117 3.0 99.6 2.6

Thiofanox 4.0 87.7 2.9 88.1 3.0 101 2.9 a

TOC = 1.49 mg/L, pH=9.0 and hardness =120 mg/L as calcium carbonate.

538-38

TABLE 13. INITIAL DEMONSTRATION OF CAPABILITY QUALITY CONTROL REQUIREMENTS

Method

Reference

Requirement

Specification and Frequency

Acceptance Criteria

Sect. 9.2.1 Initial Demonstration of

Low System Background

Analyze LRB prior to any other IDC steps and any

time a new lot of solvents, reagents, and autosampler

vials are used.

Demonstrate that all method analytes are below 1/3 the MRL

and that possible interferences do not prevent the

identification and quantification of method analytes.

Sect. 9.2.2 Initial Demonstration of

Precision (IDP)

Analyze four to seven replicate LFBs fortified near

the mid-range calibration concentration. %RSD must be <20%

Sect. 9.2.3

Initial Demonstration of

Accuracy (IDA)

Calculate mean recovery for replicates used in IDP.

Mean recovery 30% of true value

Sect. 9.2.4 Minimum Reporting Limit

(MRL) Confirmation

Fortify and analyze seven replicate LFBs at the

proposed MRL concentration. Calculate the Mean

and the Half Range (HR). Confirm that the upper

and lower limits for the Prediction Interval of Result

(Upper PIR, and Lower PIR, Sect. 9.2.4.2) meet the

recovery criteria.

Upper PIR 150%

Lower PIR 50%

Sect. 9.2.5

and 9.3.7

Quality Control Sample

(QCS)

Analyze a standard from a second source, as

part of the IDC, each time a new Analyte MEOH

PDS (Sect. 7.2.2.2) is prepared, and at least

quarterly.

Results must be within 70-130% of expected value.

Sect. 9.2.6 Detection Limit (DL)

Determination (optional)

Over a period of three days, prepare a minimum of

seven replicate LFBs fortified at a concentration

estimated to be near the DL. Analyze the replicates

through all steps of the analysis. Calculate the DL

using the equation in Sect. 9.2.6.

Data from DL replicates are not required to meet method

precision and accuracy criteria. If the DL replicates are

fortified at a low enough concentration, it is likely that they

will not meet precision and accuracy criteria for CCCs.

NOTE: Table 13 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the information has been

abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional information from the method text

is needed. In all cases, the full text of the QC in Section 9 supersedes any missing or conflicting information in this table.

538-39

TABLE 14. ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY)

Method

Reference

Requirement

Specification and Frequency

Acceptance Criteria Sect. 8.1 -

Sect. 8.4 Sample Holding Time

14 days with appropriate preservation and storage as

described in Sections 8.1-8.4.

Sample results are valid only if samples are analyzed within the sample holding

time.

Sect. 9.3.1 Laboratory Reagent

Blank (LRB)

One LRB with each analysis batch of up to 20 Field

Samples.

Demonstrate that all method analytes are below 1/3 the MRL, and confirm that

possible interferences do not prevent quantification of method analytes. If

targets exceed 1/3 the MRL or if interferences are present, results for these

subject analytes in the analysis batch are invalid.

Sect. 9.3.3 Laboratory Fortified

Blank (LFB)

LFB not required unless LFSM fails QC criteria (Sect.

9.3.5.4).

If an LFB must be analyzed due to failure of the LFSM, results of LFB analyses

must be 70-130% of the true value for each method analyte for all except the

lowest standard, which should be 50-150% of the true value.

Sect. 9.3.4 Internal Standard (IS)

Internal standards are added to all standards and

samples including QC samples. Compare IS areas to

the average IS areas in the most recent calibration.

Peak areas (or calculated concentrations) for all ISs in all injections must be

within 50% of the average peak areas calculated during the most recent

calibration. If this criterion is not met, results are labeled “suspect/IS recovery.”

Sect. 9.3.5

Laboratory Fortified

Sample Matrix

(LFSM)

Analyze one LFSM per analysis batch (20 samples or

less) fortified with method analytes at a concentration

close to but greater than the native concentration, if

known. Calculate LFSM recoveries.

See Sect. 9.3.5 for instructions on the interpretation of LFSM results.

Sect. 9.3.6

Laboratory Fortified

Sample Matrix

Duplicate (LFSMD) or

Field Duplicates (FD)

Analyze at least one FD or LFSMD with each analysis

batch (20 samples or less). An LFSMD may be

substituted for a FD when the frequency of detects are

low. Calculate RPDs.

See Sect. 9.3.6 for instructions on the interpretation of LFSMD or FD results.

Sect. 9.3.7 Quality Control

Sample (QCS)

Analyze at least quarterly or when preparing new

standards, as well as during the IDC. Results must be within 70-130% of expected value.

Sect. 10.2 Initial Calibration

Use IS calibration technique to generate linear or

quadratic calibration curves. Use at least five

standard concentrations. Check the calibration curve

as described in Sect. 10.2.7.

When each CAL standard is calculated as an unknown using the calibration

curve, the analyte results should be 70-130% of the true value for all except the

lowest standard, which should be 50-150% of the true value.

Sect. 9.3.2

and Sect.

10.3

Continuing Calibration

Check (CCC)

Verify initial calibration by analyzing a low level (at

the MRL or below) CCC prior to analyzing samples.

CCCs are then injected after every 10 samples and

after the last sample, rotating concentrations to cover

the calibrated range of the instrument.

Recovery for each analyte must be within 70-130% of the true value for all but

the lowest level of calibration. Recovery for each analyte in the lowest CAL

level CCC must be within 50-150% of the true value.

NOTE: Table 14 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the information has been abbreviated

to fit the table format, there may be issues that need additional clarification, or areas where important additional information from the method text is needed. In all

cases, the full text of the QC in Section 8-10 supersedes any missing or conflicting information in this table.

538-40

FIGURE 1. EXAMPLE CHROMATOGRAM FOR REAGENT WATER FORTIFIED WITH METHOD 538 ANALYTES

AT CONCENTRATION LEVELS INDICATED IN TABLE 7. NUMBERED PEAKS ARE IDENTIFIED IN

TABLE 3.

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